Methods for assessing transendothelial barrier integrity
An integrity and endothelial technology, applied in vascular endothelial cells, biochemical equipment and methods, pharmaceutical formulations, etc., can solve problems such as difficult to apply drug exploration, difficult to accurately reproduce, and very complex.
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example 1
[0181] Genome editing of the CLDN5 transcriptional reporter gene in hPSCs.
[0182] To assess the barrier properties of endothelial cells with surrogate markers, CLDN5 was tagged with a P2A self-cleaving peptide and GFP at the 3' end ( Figure 1a ). We designed sgRNA near the stop codon of CLDN5 and simultaneously generated a donor plasmid ( Figure 1b ) to carry a promoterless P2A-GFP sequence flanked by two homology arms (HA) with piggyBac inverted terminal repeats at each end allowing scarless excision of the resistance gene cassette sequence (ITR). Double-strand breaks caused by Cas9 and sgRNA are repaired by homologous recombination between CLDN5 and the donor template ( Figure 1c ), followed by removal of the resistance gene cassette by piggybac transposase only for excision ( Figure 1d ). Single cell clones are picked and expanded. We assessed tTK deficiency by qPCR (not shown) and identified several tTK-deficient clones. Correct GFP insertion and orientation of...
example 2
[0184] Generation and characterization of a CLDN5 reporter gene in stem cell-derived endothelial cells. Human The pluripotent stem cell line reporter line and WT line differentiated into endothelial cells and 15% to 25% GFP+ cells ( Figure 2b , depending on the clone, data for one clone are shown), no GFP+ cells were observed in the WT line. GFP+ and GFP- cells were sorted by FACS, and electronic cell-matrix impedance was judged. A 1.75-fold increase in barrier impedance was observed in GFP+ cells (3200 Ω, Figure 2c ). Next, RNA-sequencing and TMT large-scale proteomic studies were performed on both FACS-sorted GFP+ and GFP- cells (not shown), and very good correlations between significantly altered proteins and corresponding mRNAs were observed. Correlation (r=0.79, p Figure 2d ). Significant upregulation of CLDN5 at the mRNA and protein levels was confirmed ( Figure 2e ), but also confirmed significant upregulation of other tight junction proteins (OCLN) and MARVELD...
example 3
[0186] CLDN5-GFP+ EC display a high functional response across the endothelial barrier integrity. Next, gene set enrichment analysis was performed (GSEA, Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al., Proceedings of the National Academy of Sciences of the United States of America. 2005; 102( 43):15545-50.), using Hallmarks MsigDB (Liberzon A, Birger C, Thorvaldsdottir H, Ghandi M, Mesirov JP, Tamayo P. Cell systems. 2015; 1(6):417-25.) database and using Ranked lists of the products of log2FC and -log10FDR of selected GFP+ cells compared to GFP- cells (data not shown) were used for this analysis. Interestingly, enrichment of angiogenesis, TGFβ, and E2F proliferation pathways was found among downregulated genes, whereas enrichment of WNT signaling was found among upregulated genes (data not shown). Pathway enrichment analysis (Zhou Y, Wang Y, Tischfield M, Williams J, Smallwood PM, Rattner A, et al., The Journal of clinical investigation. 2014;...
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